The impact of the apoptotic pathways on cancer biology is broad because the BCL-2 family of proteins regulates nearly all steps of tumor progression, exhibits prognostic value, and can be predictive of treatment success. In the clinic, tumor cells die in response to chemotherapeutics and radiation treatment by inducing apoptosis, which has generated substantial enthusiasm for dissecting how this pathway proceeds and may be pharmacologically modulated. Apoptosis proceeds when the BCL-2 family of proteins promotes mitochondrial outer membrane permeabilization (MOMP), which triggers the release of pro-apoptotic molecules from mitochondria into the cytoplasm. Two BCL-2 family proteins, BAK and BAX, directly engage MOMP by creating proteolipid pores in the outer mitochondrial membrane. BAK/BAX-dependent MOMP is required for apoptosis, and is initiated by a BCL-2 family subset, the BH3-only proteins, e.g., BID. Our studies recently discovered that in order for BAK/BAX and BID to induce MOMP, mitochondria must functionally intersect with the sphingolipid pathway. The novel hypothesis is that mitochondrial sphingolipids confer function to the BCL-2 family, which may explain how BAK/BAX and BID specifically target mitochondria. Curiosity in the BCL-2 family-mitochondrial interactions mentioned above led to the identification that the sphingolipid pathway plays a crucial role in BCL-2 family function. We recently identified that two products within the sphingolipid pathway, sphingosine-1-PO4 and hexadecenal, directly regulate BAK and BAX activation, respectively. The goals of the current application are focused on understanding the mechanistic contribution of sphingosine-1-PO4 and hexadecenal on BCL-2 family function, MOMP and apoptosis. This project emerged following years of effort to identify cellular components necessary for MOMP, and we propose three specific aims: (1) to explore the biochemical requirements for the sphingolipid pathway on BID-induced BAK/BAX activation, (2) to elucidate the functional cooperation between BAK/BAX and the sphingolipid pathway in the contexts of cellular fate and tumorigenesis, and (3) to probe the structural relationships between BAX and hexadecenal necessary to promote MOMP and apoptosis. The scientific outcome of this proposal will uncover novel mechanisms of BCL-2 family regulation and commitment to apoptosis. These data will elucidate the functional and structural interaction(s) between BAK/BAX and the sphingolipids, and serve as the foundation for the development of BAK/BAX pharmacologic regulators in the future.